The goal of this project is to identify control mechanisms in the early stages of hematopoiesis in a mouse model system, utilizing methodology for obtaining highly enriched populations of hematopoietic stem cells (HSC) from normal mouse bone marrow tissue. By isolating these cells to separate them from the majority of more mature bone marrow cells, one can ask a variety of questions regarding the nature of HSC in the mouse. These include the identification of surface antigens expressed HSC, the functional heterogeneity of the stem cell compartment at a single cell level, and the susceptibility of HSC populations to infection by pathogenic mouse viruses and by retroviral constructs. Such viruses may be exploited as vectors to introduce new genetic material into the stem cell compartment. Further, in vitro culture systems and growth factors are being tested for the ability to propagate and induce self-renewal of the enriched HSC populations. Pursuant to the goals of this project, it is critical to develop confidence in the methodology utilized to enrich the HSC population. In particular, the enrichment method should select for all primitive HSC present in the bone marrow, and not simply a subpopulation which may or may not include the most primitive members of the HSC compartment. Our results demonstrate significant variation of HSC phenotype among different mouse strains. These variations include variable expression of two major cell surface markers of HSC in mice, Ly-6A/E and Thy-1. Fortunately, one mouse strain has been identified in which the vast majority of HSC activity can be identified and isolated by immunofluorescent staining and flow cytometry. Separation of HSC by staining with a fluorescent mitochondrial dye has allowed identification of resting and activated subsets of HSC. These isolated subsets are very similar functionally, but differ in self-renewal potential. As a result, these HSC subsets will be useful in defining in vitro assays that are capable of detecting HSC self-renewal. This will result in assays for HSC that can be applied to human tissues and which will eventually reduce the requirement for experimental animals in HSC research.